All intracellular pathogens must exit their host cells in order to disseminate and transmit to new hosts, yet this central theme has received little attention. The molecular mechanisms that control the exit of the intracellular bacterium Chlamydia from host cells are also poorly understood, and exemplify the greater gap in our understanding of this fundamental question in microbial pathogenesis. Using a novel imaging-based in vitro model, we have identified the two pathways by which Chlamydia exit cells at the end of their intracellular developmental cycle. One pathway, extrusion, is particularly innovative because of its originality and unique impact on dissemination and immune evasion. Our long-range goal is to understand the underlying molecular basis of and overall strategies used by intracellular pathogens to exit host cells. Our immediate objectives are to elucidate the molecular mechanisms of chlamydial extrusion and, for the first time, determine its role in pathogenesis in vivo. Here, we present data for the host cellular pathways engaged by Chlamydia during infection and that are required for extrusion from cells. We propose that Chlamydia specifically target these pathways via localized host-Chlamydia protein interactions on the vacuole membrane surface. The results of this work will profoundly enhance our understanding of microbial exit - a long unaddressed question in the field. Extrusion represents a new theme in host-pathogen interactions, and thus significant advances in our knowledge of this process at the mechanistic level and in the infected host should directly translate to other pathogen systems in which similar exit strategies are used, such as malarial parasites. Three key themes in chlamydial extrusion will be addressed by the following Specific Aims: (i) Identify the molecular mechanism for actin recruitment to the inclusion membrane;(ii) Define the host cytokinetic signaling networks responsible for the contraction step of extrusion;and (iii) Determine the functional role of extrusion in Chlamydia cell-to-cell spread and in vivo pathogenesis. Extrusion is a new paradigm in microbial pathogenesis. This novel exit mechanism likely plays a critical role in pathogen dissemination, transmission and immune evasion. We expect these studies to define the essential host factors involved for Chlamydia, and to illuminate the role extrusion plays in vivo. This research will have a significant impact on our understanding of an untapped area of microbiology. Finally, as new molecular targets are revealed by this work, we will also generate the tools and basis for a new therapeutic platform - leveraging microbial exit as a means of controlling infectious diseases. PUBLIC HEALTH RELEVANCE: The relevance is to gain a molecular understanding of chlamydial infections, specifically in regards to how these bacteria spread from cell-to-cell. This is a novel research question that will positively impact our knowledge of other important infectious diseases, such as malaria. An appreciation of how Chlamydiae promote their exit from host cells will enable a new spectrum of antimicrobial therapeutics that target the spread of infectious agents.